The Effect of Ca2+ Signaling on RhoGTPases During
Neuroregeneration
Jessica D. Tobin, Krista L. Todd, Ph.D., Brian J. Avery, Ph.D.
Introduction
Results
Conclusio ns
• Due to the high incidence and debilitating effects of
neurological injuries, nerve regeneration is an important field of
research.
• During neuroregeneration RhoGTPases regulate various aspects
of dendrite and axonal outgrowth and growth cone motility
through their regulation of the actin cytoskeleton.
1
• Actin polymerization is also heavily regulated by Ca
2+
signaling
2
,
however the link to RhoGTPases is not clear.
• We analyzed the effects of RhoGTPases on neurogenesis by
transfecting mouse neuroblastoma (N2a) cells with
constitutively active (CA) and dominant negative (DN) forms of
GFP-tagged RhoGTPases. Additionally, we plan to analyze the
combined effects of intracellular Ca
2+
signals and mutated forms
of RhoGTPases on neurite outgrowth during
neuroregeneration.
Methods
• Mouse neuroblastoma cells (N2a, ATCC CCL-131) transfected
with DN and CA RhoGTPases via Lipofectamine® 3000
Transfection Reagent (ThermoFisher L3000001).
• Plasmids: pEGFP-N1 (Clonetech), pcDNA3-EGFP-RhoA-Q63L,
pcDNA3-EGFP-RhoA-T19N, pcDNA3-EGFP-Rac1-Q61L, pcDNA3-
EGFP-Rac1-T17N, pcDNA3-EGFP-Cdc42-Q61L (Addgene
plasmids: 12968, 12967, 12981, 12982, 12986,).
3
• Plasmids prepared with ZymoPure Plasmid Midiprep Kit
(Zymogen: Catalog Nos. D4200 & D4201).
• Calcium ionophore A23187 (Sigma C7522) utilized to increase
intracellullar Ca
2+
concentration .
• Images obtained on a Zeiss Axio Examiner with a 20X water
immersion lens with AxioVision software.
Figure 2: Average number of protrusions \ cell, examined
from mixed transfected and non-transfected cells.
Figure 3: Average number of protrusions \ cell,
examined from transfected cells only.
Figure 4: Average length of protrusions \ cell in μm,
examined from transfected cells only.
Figure 1: Representative bright-field and fluorescence images of RhoGTPase transfected N2a
cells.
References
1) Hall, A., & Lalli, G. (2010). Rho and Ras GTPases in axon growth, guidance, and
branching. Cold Spring Harbor perspectives in biology, 2(2), a001818.
2) Aspenström, P. (2004). Integration of signalling pathways regulated by small GTPases
and calcium. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1742(1), 51-
58.
3) Subauste, M. C., Von Herrath, M., Benard, V., Chamberlain, C. E., Chuang, T. H., Chu,
K., ... & Hahn, K. M. (2000). Rho family proteins modulate rapid apoptosis induced by
cytotoxic T lymphocytes and Fas. Journal of Biological Chemistry, 275(13), 9725-9733.
Hypotheses
• We hypothesized that the following plasmid transfections of
RhoGTPases would affect the average number of protrusions
per cell.
• CA Rac1 and CA RhoA: increase the average number of
protrusions per cell
• CA Cdc42: decrease the average number of protrusions per
cell
• We hypothesized that the addition of calcium ionophore
A23187 would also increase the average number of protrusions
per cell.
• Overexpression of Cdc42-CA, RhoA-CA, and Rac1-CA resulted in distinct
morphological changes in N2a cells.
• Cdc42-CA overexpression induced lamellipoidal-type extensions.
• RhoA-CA overexpression increased length of protrusions.
• Rac1-CA overexpression increased number of protrusions.
• Calcium ionophore experiments are ongoing.
